PROJECT SUMMARY/ABSTRACT The potential to model the human body on a microchip offers tantalizing hope of predictive drug testing and unprecedented control for mechanistic experiments. However, existing organ-on-chip systems exclude the lymph node (LN), the small and highly organized organ that initiates adaptive immune responses. Without a LN, the induction and development of antibody- or cell-mediated immunity is also largely absent. Other available in vitro LN-mimetic systems do not yet address the crucial spatial organization and local microenvironment of this tissue. As most humans want to keep their LNs, an experimentally tractable, biomimetic model of the dynamics and organization of this organ is needed both for mechanistic studies and to test new therapies. In this project, our uniquely qualified team of engineers and immunologists will develop and validate the first spatially organized, 3D-cultured microphysiological model of a lymph node (LN-chip), featuring biomimetic cellular organization and fluid flow. In Aim 1, we will establish methods to micropattern primary human immune cells in 3D culture inside a microfluidic chip, using on-chip photolithography of photo- crosslinkable gels. This innovative approach provides simultaneous control over cellular distribution, local matrix composition, and fluid flow, to replicate diffusion and migration distances for 3D cell-cell interactions. We will optimize patterning and culture conditions to maintain viability for 7 ? 28 days, preserve T and B cell response to simple stimuli, and test multiple materials for the microfluidic housing. In Aim 2, we will identify the best strategy to achieve biomimetic lymph node organization by comparing the robustness of microstructure obtained by patterning chemokine gradients, stromal/endothelial cells, or lymphocytes. We will also determine the optimal fluid flow conditions for biomimetic function. In Aim 3, we will establish conditions for productive T-B cell interactions on the LN-chip leading to differentiation and production of long-lived, high-affinity antibodies. Responses on the LN-chip will be directly compared to those of ex vivo cultured human tonsils, to provide definitive data on the relevance of the model to human immunity. Finally, we will employ CRISPR/Cas9 gene editing to test the extent to which the LN-chip recapitulates human disease caused by defects in T?B interaction. In summary, this U01 project will produce validated procedures for robust and reproducible assembly of the first spatially organized LN-chip, including specific guidelines for inclusion of stromal cells and lymphocytes, and benchmarking against well-defined human T- B interactions. The platform will be broadly applicable to model inflammatory and autoimmune diseases, test vaccination strategies, and answer mechanistic questions about LN function. It will be compatible with in-line coupling to other organs-on-chip from the Tissue Chip consortium, and will allow for direct testing of patient lymphocyte function within a model tissue microenvironment, ultimately enabling both small molecule and CRISPR/CAS9 genetic based screens.